Receptors for IgM and IgM-antigen complexes on human T-lymphocytes reacting with specific anti-human-T-cell antiserum.

A receptor on T lymphocytes that can bind IgM-type antibodies, without, as well as after prior complex formation between the antibody and Esh (sheep red blood cells) has been detected. No prior in vitro incubation of freshly drawn and isolated peripheral blood lymphocytes is required for the expression of the IgM antibody binding receptor on T cells. Results obtained in experiments designed for the simultaneous detection of lymphocytes reacting with specific rhodamine labelled antihuman-T cell antiserum and forming rosettes with EA-IgM complexes of Esh and the IgM antiserum fraction showed the receptor for IgM antibody to be present exclusively on T cells. Pre-incubation of T cells with free IgM or its Fc fragments leads to inhibition of rosette formation with EA-IgM. Treatment of the IgM type pentameric antibody with dithioerythritol to cleave pentameric IgM reduces the percentage of EA-IgM rosette forming cells (RFC) significantly. The possible biological significance of this receptor is discussed.     

Receptors for IgM on Human B Lymphocytes

Using a rosette technique with IgM coated bovine red blood cells (EA-IgM) receptors for IgM can be demonstrated on human B-lymphocytes. While in the peripheral blood B cells with IgM receptors are found only occasionally, between 7 and 33%, mean 16%, of tonsil B-lymphocytes exhibit receptors for IgM. This was shown in double marker studies using EA-IgM for the demonstration of IgM receptors and fluorochrome labelled conjugates for the demonstration of S-IgD, S-IgM and B cell antigens. These receptors are specific for IgM, they can be completely blocked by IgM-anti OVA complexes and partially by free IgM, but not at all by aggregated human IgG. They are sensitive to trypsin and pronase but reconstitute after further incubation at 37 degrees C. These data show that not only T and CLL cells but also some normal B-lymphocytes have receptors for IgM. We favour the view that CLL lymphocytes may derive from these B-lymphocytes, which may represent a certain maturation step in B cell development.     

Subpopulations of Human Lymphoblastoid Cell Lines

Forty lymphoblast cell lines derived from normal subjects, patients with infectious mononucleosis, leukemia, and Burkitt's lymphoma have been studied for surface receptors including surface Ig, complement receptors by the EAC rosette and fluorescent (Raji cell) techniques, and Fc (aggregate) receptor by direct and indirect immunofluorescence. Because of the B-cell tropism of the Epstein-Barr virus (EBV), an effort was made to correlate the expresion of various surface properties of lymphoblastoid cell lines with the content of EBV viral DNA as determined by complementary RNA-DNA (cNRA-DNA) hybridization on membrane filters or by DNA-DNA renaturation kinetic analysis. The only correlation established was with the Fc receptor determined by direct immunofluorescence. No correlation of EBV genome equivalents per cell with complement receptor or surface Ig was noted, suggesting that the expression of these receptors is not influenced by EBV viral DNA content. Subgroups of lymphoblastoid cell lines were on the basis of variable expression of surface receptors, designated B1, B2, B3, B4, and T. The distribution of lymphoblastoid cell lines into these subgroups were in the ratio of 14:4:1:4:1. The B1, B2, and B4 cell lines (except Molt 4F) were found to contain EBV. The B3 subgroup, for wich cell line 698 was the sole example, expressed surface immunoglobulins but no other B-cell characteristics, and H.S.B., a T-cell line, lacked detectable EBV.     

Isolation of various canine leucocytes and their characterization by surface marker analysis.

Various techniques were used to separate canine peripheral blood leucocytes into populations enriched in lymphocytes, polymorphonuclear leucocytes, phagocytic mononuclear cells (monocytes) and macrophages. Surface markers on each cell population were determined by rosette formation. Fc receptors for IgG and complement receptors (C3b and C3d) were present on PMN, monocytes, macrophages as well as on a sub-population of lymphocytes. Purification of the lymphocytes into T-and B-cell-enriched populations revealed that these receptors were present only on the B lymphocytes and not on the T lymphocytes. In addition, a third lymphocyte population, which did not possess surface immunoglobulin, and Fc receptor but not the complement receptor. None of the cell populations exhibited C4 complement receptors or Fc receptors for IgM. When different cell populations were tested for their ability to form rosettes directly with human type 'O' red blood cells it was found that most populations could rosette, suggesting that this technique could not be used as a specific marker for canine T lymphocytes.     

The functional activity of Fc gamma RII and Fc gamma RIII on subsets of human lymphocytes.

Subsets of human lymphocytes were isolated from peripheral blood using magnetic beads coated with anti-CD4, -CD8, -CD19 or -CD56 antibodies to yield T4, T8, B and natural killer (NK) cell suspensions with greater than 95% purity. The functional activity of Fc gamma receptor II (Fc gamma RII) and Fc gamma receptor III (Fc gamma RIII) on these subsets was assessed by measuring rosette formation with red cells sensitized with known levels of either rabbit IgG or human (monoclonal or polyclonal) IgG1 anti-D, IgG3 anti-D or IgG3 anti-c (E-IgG). Lysis of red cells by K cells (mediated by Fc gamma RIII) in antibody-dependent cell-mediated cytotoxicity (ADCC) assays was promoted by polyclonal and some monoclonal antibodies. Using these 'ADCC+' antibodies, minimum red cell sensitization levels required to promote rosette formation with NK cells were 2000 IgG1 or IgG3 molecules/red cell compared to 15,000 IgG1 or 4000 IgG3 molecules/red cell with 'ADCC-' monoclonal antibodies. The greater efficiency of ADCC+ antibodies is consistent with their previously reported ability to bind Fc gamma RIII via CH2 and CH3 domains whereas ADCC- antibodies bind only via CH3 domains. B cells formed rosettes only at high levels of sensitization: approximately 60,000 IgG1 or 20,000 IgG3 anti-D molecules/cell. These data reflect the low affinity of Fc gamma RII for monomeric human IgG. Although over 90% of NK cells bound anti-CD16, and 70% formed rosettes with red cells sensitized with rabbit IgG (30,000 molecules/cell), only 25% of NK cells formed rosettes with E-IgG3 at 100,000 IgG molecules/cell. Approximately 35% of B cells, 10% of T8 cells but no T4 cells formed rosettes with E-IgG (100,000 IgG3 molecules/cell). With T8, B and NK cells, IgG3 anti-D promoted greater rosette formation than IgG1 anti-D at comparable levels of sensitization. Presumably the longer hinge region of IgG3 enabled it to bridge the gap between negatively charged lymphocytes and red cells more efficiently than IgG1.     

Functional and clinical consequences of Fc receptor polymorphic and copy number variants

Receptors for immunoglobulins (Fc receptors) play a central role during an immune response, as they mediate the specific recognition of antigens of almost infinite diversity by leucocytes, thereby linking the humoral and cellular components of immunity. Indeed, engagement of Fc receptors by immunoglobulins initiates a range of immunoregulatory processes that might also play a role in disease pathogenesis. In the circulation, five main types of immunoglobulins (Ig) exist – namely IgG, IgA, IgE, IgM and IgD and receptors with the ability to recognize and bind to IgG (Fcγ receptor family), IgE (FcεRI and CD23), IgA (CD89; Fcα/µR) and IgM (Fcα/µR) have been identified and characterized. However, it is astonishing that nearly all the known human Fc receptors display extensive genetic variation with clear implications for their function, thus representing a substantial genetic risk factor for the pathogenesis of a range of chronic inflammatory disorders.     

T-cell hybridoma co-expressing Fc receptors for different isotypes. I. Reciprocal regulation of Fc alpha R and Fc gamma R expression by IgA and interferon.

To clarify the co-expression phenomenon of T-cell Fc receptors (FcR) specific for different isotypes on the clonal level, a murine hybridoma clone T2D4 was studied. T2D4 cells originally reported to bear FcR for IgG (Fc gamma R) and to release a Fc gamma R-related T-cell factor binding to IgG (immunoglobulin binding factor; IBF) proved to have also the receptor for IgA. The binding of IgA was detected by rosette formation with trinitrophenylated ox red blood cells (TNP-ORBC) after preincubation of T2D4 cells with MOPC 315 IgA having anti-TNP activity, or directly with TNP-ORBC sensitized with MOPC 315 IgA. While the binding of MOPC 315 IgA was competed for by IgA but not by IgG2A nor IgG2B, IgA failed to inhibit the rosette formation of the cells with ORBC sensitized with rabbit IgG antibody (EA ox gamma), proving that T2D4 cells express FcR specific for IgA (Fc alpha R) in addition to Fc gamma R. Co-expression of both receptors on the same cell surface was demonstrated by a double rosette technique using TNP-quail red blood cells (TNP-QRBC) and EAox gamma. Fc alpha R activity of the cells was completely abrogated by 15 min. incubation with 0.1 mg/ml trypsin, whereas Fc gamma R was resistant even to 1 mg/ml trypsin. The expression of Fc alpha R was augmented (up-regulation) by IgA at the concentration above 300 micrograms/ml and inhibited (down-regulation) by 1000 u./ml of murine beta-interferon (beta-IFN). Conversely, the expression of Fc gamma R was down-regulated by IgA and up-regulated by alpha-IFN. Thus, Fc gamma R and Fc alpha R are co-expressed and reciprocally regulated on these cell lines. The possible co-production of IBF and the Fc alpha R-related binding factor specific for IgA is discussed.     

Human monoclonal antibodies produced by Epstein-Barr virus transformed cell lines bind protein A

Epstein-Barr virus (EBV) is a polyclonal T-independent activator of viral receptor positive human B lymphocytes. Lymphocytes infected in vitro with the virus are transformed into immortalized cell lines [Nilsson, K, and Klein, G. (1982) Adv. Cancer Res. 37, 319]. In this way human cell lines that secrete specific IgM, IgG and IgA monoclonal antibodies are established. Protein A is also a polyclonal T-independent B cell activator [Langone, J. J. (1982) Adv. Immunol. 32, 157], the targets of which are surface immunoglobulin and C3d receptor positive cells, as are the targets of EBV. We found that almost all (16 out of 17) of the specific monoclonal antibodies (IgM, IgG and IgA) produced in vitro by EBV cell lines bind protein A. Unlike these in vitro produced antibodies, a substantial fraction of the immunoglobulins in human serum does not bind protein A. Thus, those plasma cells which in vivo secrete protein A nonbinding immunoglobulins originate from precursors of B cell that were EBV noninfective. Alternatively, during in vivo B differentiation some immunoglobulins undergo a change from protein A binding to protein A nonbinding molecules.     

Fc receptors on rabbit lymphocytes Existence of receptors for IgG antibody complexed with antigen; conditions for its detection

The presence of Fc receptors (FcR) on rabbit peripheral blood leukocytes is demonstrated using rosette formation with an ox erythrocyte-antibody (EoxA) complex. The receptor is specific for the Fc fragment of IgG (neither IgM nor F (ab′)₂ anti-Eox mediates rosette formation) that is antigen-bound (aggregated rabbit IgG inhibits the rosette formation only transiently). The receptor is species-specific: guinea pig IgG/ Eox, goat IgG/Eox and sheep IgG/Eox complexes do not show rosette formation, and goat IgG aggregates do not inhibit rosette formation. The origin of the target erythrocytes is of importance. Sheep erythrocytes are not useful, and within Eox large differences between donors were found. Rosette formation was only inhibited by pretreatment of the rosette-forming cells with homologous immune complexes, whereas the size of the antigen greatly influenced the degree of inhibition. The rabbit FcR is pronase-resistant, unlike the human and murine FcR. The interaction of IgG and the FcR is not inhibited by isolated Cγ 3 domains. Further evidence for the requirement of the whole Fc region was obtained in experiments where inhibition of the rosette formation was observed using antisera directed to the Cγ3 and the Cγ2 domain, respectively. Anti-Fab antiserum did not inhibit rosette formation. Results are discussed in relation to the mechanism of allotypic suppression.     

Isolation and Characterization of Fc Receptors from Haemophilus somnus

Receptors that bind the Fc region of bovine immunoglobulin (Ig) have been isolated from the culture supernatant of Haemophilus somnus by chromatography on a Sepharose 4B column. One receptor with a relative molecular weight of 41,000 weakly binds both bovine IgG subclasses, IgA and IgM, while three high molecular weight receptors (350,000, 270,000, and 120,000) strongly bind bovine IgG2, IgA, and IgM. All four Fc receptors are antigenically related and the 41,000 receptor appears to be a subunit of the high molecular weight receptors. In addition to bovine Ig, the purified 270,000 Fc receptor strongly binds horse IgG, rabbit IgG, pig IgG, cat IgG, dog IgG, and sheep IgG. The receptor also reacts weakly with mouse, rat, chicken, human, and guinea pig IgG and does not bind goat IgG. Fc receptors from 19 H. somnus isolates were compared. Variations in the molecular weight of the 41,000 protein were demonstrated among preputial isolates from asymptomatic carriers, but all other isolates appeared to have identically migrating proteins.     

Antibody-dependent cellular cytotoxicity mediated by subpopulations of human T lymphocytes: killing of human erythrocytes and autologous lymphoid cells.

The present study characterized the cytotoxic capabilities of human T lymphocyte subpopulations against human red blood cells (HRBC) and autologous lymphoid cells in an antibody-dependent cellular cytotoxicity (ADCC) assay. T cells bearing Fc receptors for immunoglobulin IgG (TG) were capable of lysis of antibody-coated HRBC and autologous lymphoid cells while T cells with surface Fc receptors for IgM (TM) displayed no ADCC activity TG-cell mediated ADCC could be inhibited by blockage of surface Fc receptors following treatment with aggregated Ig. Null cells and low-affinity E-rosette forming cells were also capable of similar ADCC activity against these targets.     

Increased 'T lymphocyte' bearing Fc receptors for IgG in pregnancy

T cell subpopulations as defined by E rosette formation and Fc receptors for immunoglobulins were determined, using ox red blood cells coated with the IgG or IgM fraction of rabbit anti-ox red blood cells antibody to form rosettes with the peripheral blood lymphocytes of 18 pregnant females and 12 healthy nonpregnant females. It was shown that the TG cell population in the pregnant females is significantly increased as compared to those in the nonpregnant controls (mean +/- SEM % TG cells: 18 +/- 1.2 vs. 9.6 +/- 0.7; p less than 0.001). By using peripheral blood from normal nonpregnant subjects it was also shown that TG cells suppressed one-way mixed lymphocyte reactions (mean +/- SEM suppression: 23 +/- 7.2; p less than 0.01). These findings suggest that the TG cell population may exert a suppressor function on the immune response to alloantigens and act in concert with other humoral factors to protect the fetus from rejection.     

Specificity of Receptors for IgG on Human Lymphocyte-Like Cells

Some human lymphocyte-like cells (EA-RFC) have receptors for IgG demonstrable by their ability to form rosettes with human Rh-positive O erythrocytes sensitized with anti-CD isoantibodies (Ripley). The specificity of these receptors for the various Ig classes, IgG subclasses, and fragments of the IgG molecule was determined by studying the inhibitory capacity of the corresponding immunoglobulins in the rosette assay. The receptors showed specificity only for IgG among the Ig classes and about equal affinity for IgG1 and IgG3, but only weak binding of IgG2 and IgG4 was obtained. Whereas no inhibition was obtained with Fab and F(ab')₂ fragments prepared from IgG, the Fc fragment showed strong inhibitory capacity, which was even surpassed by the IgG3 Fch fragment, containing an extension from the N-terminal part of Fc. The inhibitory capacity of the Fc and Fch fragments was considerably reduced by partial reduction and alkylation. The pFc' fragment of IgG, which corresponds to the C_γ3 region, did not inhibit rosette formation. These data indicate that mainly the C_γ2 region is involved in the binding of IgG to EA-RFC. Inhibition studies did not show any differences in the relative inhibitory capacity of monomerie, dimeric, or highly polymerized (heat-aggregated) IgG. However, antibodies of rabbit origin complexed with antigen (ferritin) gave stronger inhibition than the corresponding native Ig.     

Expression of a receptor for IgM by human T cells in vitro

The capacity of human peripheral blood lymphocytes (PBL) to bind antigen-IgG antibody (EA(IgG)) and antigen-IgM antibody (EA(IgM)) complexes was investigated using a rosette technique with ox erythrocytes coated with rabbit IgG or IgM antibody. It was found that while EA(IgG)-rosette-forming cells (RFC) were detected on PBL freshly drawn from normal individuals, EA(IgM)-RFC were present only in suspensions kept in culture for 24 h in mediá supplemented with sera containing very low or no amounts of IgM. Experiments of simultaneous detection of EA(IgG)-RFC or EA(IgM)-RFC and other membrane markers for human T or B cells together with experiments on purified T or B cell populations indicated that EA(IgG)-RFC were formed by both T and B cells, while T cells only were capable of EA(IgM) rosette formation. The specificity of the receptors for IgG and IgM was determined by studying the inhibitory capacity of purified human IgM and IgG in the rosette assay. The receptor for IgG was inhibited by IgG and not by IgM, while the reverse was true for the receptor for IgM.     

Induction of Fc receptors for IgA on murine T cell hybridoma by human monoclonal IgA and by high molecular weight IgA in IgA nephropathy.

A reproducible immunocyto-adherence assay has been developed to study the modulation of Fc receptors for IgA (Fc alpha R), using a murine T cell hybridoma (T2D4), which expresses Fc receptors for all known isotypes of secreted immunoglobulins. By using sheep red blood cells coated with the hapten 2-4-6 trinitrophenyl (TNP), as indicator cells, and a murine monoclonal IgA (MOPC 315) antibody with anti-TNP activity, we were able to study the Fc alpha R on T2D4 cells. We found that: (a) murine Fc alpha R can bind human monoclonal IgA, and this binding is isotype specific since it was inhibited by human monoclonal IgA but not by human monoclonal IgG or IgM; (b) the expression of murine Fc alpha R is unducible by human monoclonal IgA, and this effect is isotype specific since it is not observed with human monoclonal IgM or IgG (c) sera from patients with IgA nephropathy can also induce Fc alpha R expression; by contrast, no induction was observed with normal human sera, (d) in one serum from an IgA-nephropathy patient, the inducer factor was characterized by affinity chromatography on anti-IgA-Sepharose and by gel filtration: high molecular weight IgA, probably IgA aggregates or immune complexes were recognized to be responsible for the induction of murine Fc alpha R expression.     

Phagocytic peripheral blood monocytes from rabbits and humans express membrane receptors specific for IgM molecules: evidence that incubation with neuraminidase exposes cryptic IgM (Fc) receptors.

Phagocytic human and rabbit peripheral blood monocytes, identified by their ingestion of polystyrene particles, were investigated for the presence of surface membrane receptors for IgM molecules. After incubation of freshly isolated monocytes with IgM anti-sheep erythrocyte (SRBC) preparations, a mean of 0.7% of human monocytes and a mean of 16.2% of rabbit monocytes formed rosettes with SRBC. However, if the monocytes were pre-incubated with vibrio cholerae neuraminidase (VCN), these figures increased to 32.6% and 37.8% respectively. The specificity of rosette formation by VCN-treated monocytes was established in several experiments; SRBC sensitized with a F(ab')2 preparation of an IgG anti-SRBC reagent completely failed to rosette with VCN-treated monocytes, and inclusion of IgM, but not other Ig or non-Ig protein molecules in the test medium, inhibited rosette formation. Further, and most important, rosette formation by human monocytes was inhibited by F(c)5mu but not by Fabmi fragments. These findings indicate that both rabbit and human monocytes express IgM-class specific membrane receptors for IgM molecules, that these receptors may be cryptic or hidden but can be revealed by treatment with VCN and that the human monocyte IgM receptor is F(c) specific. Further, the rabbit monocyte IgM receptor was shown to be trypsin-resistant.     

Neuraminidase- and trypsin-induced exposure to membrane receptors for IgG and IgM molecules on human peripheral blood lymphocytes.

Brief incubation of human peripheral blood lymphocytes with vibrio cholerae neuraminidase (VCN) or trypsin revealed hidden membrane receptors for IgG and IgM molecules. The hidden receptors were found on T lymphocytes as shown by double-label and mixed rosetting experiments and by studies of T-enriched populations. Although IgM receptors were undetectable on freshly isolated lymphocytes, a mean of 17.1% of VCN-treated lymphocytes rosetted with ox erythrocytes coated with IgM antibody (EA-IgM). Prior to trypsin treatment a mean of 14.6% of human T lymphocytes rosetted with ox red cells coated with IgG antibody (EA-IgG). After incubation with trypsin this figure increased significantly (P less than 0.005) to 44.5%. VCN-treatment also significantly increased (P less than 0.005) the mean percentage of EA-IgG rosette-forming T cells to 38.5%. The T-cell receptor for IgG was shown to be trypsin-resistant while the IgM receptor was shown to be trypsin-sensitive. Using mixed rosettes, a tentative T-cell subset was identified which expressed both IgG and IgM membrane receptors. Also, a minor subset with IgM receptors alone and a larger subset with only IgG receptors were identified.     

Characterization of K-cell activity by use of depletion experiments.

Normal human blood lymphocytes with affinity for ox red cells sensitized with IgG antibody, for normal or papain-treated sheep red cells and for ox red cells coated with mouse complement were used for rosette formation and the rosetting cells separated by density gradient centrifugation on Ficoll/hypaque. The non-rosetting cells at the interface were collected and compared with cell suspensions before treatment for direct and antibody-dependent cytotoxicity of human target cells. Depletion of Fc-receptor-bearing lymphocytes strongly decreased antibody-dependent cell-mediated cytotoxicity; reduction in the number (or depletion) of T cells and cells with C'3 receptor had no effect, showing the same or enhanced K-cell activity. It is concluded that one type of K or killer cell has Fc receptors but lacks C'3 receptors.     

Cellular requirements for the formation of EA rosettes by human monocytes.

The binding of sensitized red cells to Fc receptors in human monocytes was studied by evaluating the effects of various pharmacological reagents and other treatments on EA rosette formation. Cytochalasin B and 2-deoxyglucose inhibited rosette formation in a dose-dependent manner. Sodium azide and incubation at 4 degrees also inhibited rosette formation, while at 37 degrees increased numbers of RBCs bound to the monocytes. The microtubular poisons, vinblastine and colchicine at high concentrations resulted in decreased adherence of monocytes and inhibition of rosette formation, while at low concentrations of colchicine, enhanced rosette formation was sometimes observed. Contrary to the effects on rosette formation, binding of [125I] IgG to monocyte monolayers was not altered by treatment of the monocytes with drugs. Magnesium ions were required to promote monocyte adherence, but both magnesium and calcium were needed for the best rosette formation. We conclude that the formation of EA rosettes is dependent not merely on binding of IgG to the Fc receptor but requires metabolically active monocytes, an intact cytostructure and suitable environmental conditions (temperature and cation concentration).     

Sheep erythrocyte rosette formation by B chronic lymphocytic leukemia cells as a consequence of monoclonal surface immunoglobulin with "Forssman-like" antibody activity

Peripheral blood and splenic lymphocytes from an elderly man with chronic lymphocytic leukemia (Rai stage 4) were shown to have monoclonal surface immunoglobulin (IgM+, IgD+, kappa+), Ia-like antigen and receptors for unsensitized sheep red blood cells. The sheep red blood cell receptor was not blocked by monoclonal antibodies that bind to the classic T lymphocyte rosette receptor (OKT-11 and Lyt-3) or by anti-human IgG or antilambda antibodies. However, the sheep red blood cell receptor was blocked by antihuman IgM and kappa antisera and by soluble guinea pig kidney antigen (Forssman antigen). It is concluded from these and other observations that our patient has a B-cell lymphoproliferative disorder expressing monoclonal surface immunoglobulin with anti-"Forssman-like" antibody activity.